MS analysis of photocrosslink adducts for the mapping of interaction sites of selective engineered PSD-95 PDZ domain ligands

ABSTRACT: Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces and PPI domains. We report here a strategy that addresses this challenge by using a semi-rational approach that separates the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by employing a selection by phage display coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to bind against the target interface. We have applied this approach to PSD-95, an essential multi-PDZ domain-containing synaptic scaffold protein that belongs to a larger family of paralogs. We show here that with this strategy we could specifically target a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides the first paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules. This archive contains the mapping by photocrosslinking and LC/MS/MS analysis of the interaction sites of engineered selective PSD-95 PDZ domain ligands, Xph20-ETWV, Xph20-Stg and Xph20-ETMA.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

SUBMITTER: Stephane Claverol  

LAB HEAD: Marc Bonneu

PROVIDER: PXD015366 | Pride | 2019-11-12

REPOSITORIES: Pride